ABSTRACT Ample data support a role for impaired lysosomal acidification and reduced autophagic flux in comorbidities related to obesity, including non-alcoholic fatty liver disease (NAFLD), heart failure, and impaired insulin secretion in diabetes. Investigation of the role of impaired lysosomal acidification in the pathophysiology of NAFLD, heart failure, and diabetes has been hampered by the lack of a technology to acidify lysosomes. To facilitate mechanistic studies of acutely restoring lysosomal pH, we recently published that photoactivated release of acid from lysosome-targeted nanoparticles (NPs) provides immediate short-term restoration of optimal pH and autophagic flux. However, to translate such technology for long-term acidification and for in vivo use, there is a need to develop NPs that release acid independent of an external trigger. This proposal stems from our development of novel formulations that allow lysosome-targeted NPs to sense their entry into the lysosomes and then trigger the release of acid. To this end, we capitalized on the observation that impaired acidification, documented in various diseases, only elevates the pH by 0.6-1 units. Therefore, the pH of the dysfunctional lysosome is still significantly more acidic than the cytosol and plasma. Consequently, we designed a new NP formulation that releases acid at pH 6 and below to assure that acid release will only occur in the lysosome. We hypothesize that acid-activated-acid-release NPs (acNPs) will allow for continuous treatment and long-term restoration of lysosomal acidity and autophagic flux. In this study we will develop, test, and validate acNP in cultured cells. Our long term goal is to develop the acNPs to be used in vivo. Preliminary data show that the acNPs efficiently target lysosomes. Furthermore, preliminary data in a hepatocyte cell line demonstrate the capacity of the acNPs to restore pH and autophagic flux under conditions of impaired lysosomal function induced by chronic exposure to excess lipids. To address our hypothesis we propose the following two aims: Aim1: Synthesize and characterize lysosome targeted acNPs that activate at pH 6 where we will test the hypothesis that lysosome targeted acNPs enter the cells through endocytosis and follow them through their maturation into the lysosome, where at pH ?6 they will trigger acid release and restore lysosomal pH and autophagic flux. And Aim 2: Determine the capacity of acNPs to enable long-term restoration of autophagic flux under conditions that impair lysosomal acidification where we will test the hypothesis that in cells treated with excess lipid environment, re-acidification of lysosomes by acNP will restore autophagic flux, mitochondrial turnover, and cellular functions including insulin sensitivity in the hepatocyte and insulin secretion in the beta cell.